Over the past five decades, the neuroscientific community has made significant progress in our understanding of the organization and function of the spinal cord dorsal horn and dorsal root ganglia (DRG). Many new proteins have been identified as important in the pain signal conduction pathway. Novel gene therapy approaches may offer new opportunities for long-term pain management strategies. The adeno-associated virus (AAV) serotype has been shown to be useful for transduction of neurons in cortex, brainstem, and cerebellum. Its utility for transduction of neurons in the spinal cord and dorsal root ganglia has also been explored; attempts to deliver the AAV vectors to the spinal cord or DRG have only been successful with direct intraparenchymal or intraneural injections. Less invasive and more clinically therapeutic direct lumbar puncture approaches have not successfully transduced the spinal cord. In contrast, we have now succeeded in achieving widespread spinal cord and DRG transduction of the marker green fluorescent protein (GFP) by direct lumbar puncture injection of an AAV serotype 5 (AAV5) in conscious mouse and rat. These preliminary studies support our proposal to optimize delivery and characterize potential function of AAV5 based vectors targeting specific genes known to modulate chronic pain signaling within spinal cord or DRG. Efficient AAV5-mediated genetic manipulation offers substantial opportunities to 1) further study mechanisms underlying chronic pain and 2) develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. The proposed research will assess the utility of delivery of AAV-vector by lumbar puncture as a useful tool for basic scientific study of chronic pain as well as a potential therapeutic delivery option. The primary objectives of the project are: 1) To optimize delivery to the spinal cord and DRG and characterize the distribution of the AAV5-GFP construct. 2) To validate this approach, in a system well established in the pain signal conduction pathway. 3) To apply the approach to a novel non-opioid system that may exert control on the development of chronic opioid tolerance and maintenance of chronic pain. Future applications of intrathecal delivery of AAV5 constructs will enhance study of other novel targets participating in chronic pain at the level of the spinal cord and DRG and may enable translational developments of chronic pain therapies. [unreadable] [unreadable] [unreadable]